PROJECT SUMMARY/ABSTRACT S-adenosylmethionine (SAM) is the methyl donor for nearly all cellular methylation reactions. Given the importance of methylation in the activities of nucleic acids, proteins, and lipids, proper control of SAM is fundamental to a wide array all of cellular processes. Nonetheless, the mechanisms cells use to maintain SAM homeostasis remains incompletely understood. Recent data suggest a model in which a posttranscriptional feedback loop regulates SAM levels. The model proposes that cells respond to intracellular SAM levels by controlling intron retention of MAT2A, the RNA that encodes the major SAM synthetase. A conserved hairpin, called hp1, in the MAT2A 3 UTR is a key cis-acting determinant in the response and the recently characterized RNA methyltransferase METTL16 is also central to the pathway. Under high SAM levels, METTL16 methylates MAT2A hp1 favoring intron retention and nuclear degradation of the MAT2A transcript. Upon SAM depletion, METTL16 binds hp1 but stays bound, presumably due to poor enzymatic turnover in low SAM. When bound to the RNA, METTL16 functions as a splicing enhancer to promote MAT2A mRNA production thereby increasing SAM synthetase and SAM levels. Despite significant empirical support for this model, it only begins to define the mechanisms and biological significance of METTL16 and posttranscriptional regulation of SAM homeostasis. The three aims in this proposal seek to further understand the biological roles of METTL16 controlling SAM homeostasis and RNA processing. In Aim 1, the cis- and trans-acting factors required for splicing will be defined to elucidate the mechanism of METTL16-induced splicing. In Aim 2, the proposed links between SAM homeostasis and MAT2A regulation by METTL16 are tested. Finally, in Aim 3, an unbiased screen is described that has identified potential new regulators of the SAM homeostasis in cells. Because SAM is central to many cellular functions, loss of precise control of SAM levels has potentially serious consequences for human health. The work described here will define mechanisms cells use to maintain SAM homeostasis.